Author(s): ENGMAN, V., CRITCHLOW, A.J., WILLIAMS, R.M., VAN BELLEGHEM, K., GARNHAM, A., MASON, S., LAMON, S., Institution: DEAKIN UNIVERSITY, Country: AUSTRALIA, Abstract-ID: 1239

INTRODUCTION:
Plasma concentrations of androgens and oestrogens are moderated by sex and naturally fluctuate across the lifespan. Recent evidence suggests that sex hormones can also be synthesised locally in skeletal muscle. Previous research is however limited by small sample sizes and the use of immunoassays, which may yield unreliable results at low hormonal concentrations. To overcome these limitations, we developed a new liquid chromatography-mass spectrometry (LC-MS)-based technique to quantify intramuscular hormone concentrations. Using this method, we quantified the intramuscular sex hormone concentrations in muscle biopsies from a sample of 134 healthy males and females representing each decade of adulthood.
METHODS:
A fasting muscle biopsy was obtained from the vastus lateralis and snap frozen in liquid nitrogen, and a fasting venous blood sample was also collected. 2H or 13C labelled internal standards for the relevant androgens, oestrogens, and progestogens were added to ~25 mg of muscle tissue and 200 µL of plasma. The steroids were extracted and derivatised and then analysed on a Vanquish Flex LC coupled to an Orbitrap Exploris 240 mass spectrometer. The muscle was also stained for fibre-typing.
RESULTS:
Intramuscular hormones were quantified in 48 males and 86 females aged 18–80 years. Intramuscular DHEA and pregnenolone concentrations decreased significantly with age in both sexes, whereas intramuscular testosterone only decreased with age in males, but not females. DHT levels remained unchanged in both males and females with ageing. Total plasma and intramuscular DHEA concentrations showed a strong correlation with each other in males (r=0.8) and females (r=0.9). Pregnenolone showed moderate correlations in males (r=0.5) and strong correlations in females (r=0.8). Testosterone showed strong correlations in males (r=0.7) and moderate correlations in females (r=0.5). No significant correlations between plasma and intramuscular DHT were found in either group (p>0.05). Sex-specific linear regression analyses, controlling for age and plasma hormone concentrations, found no independent association of intramuscular DHEA, testosterone, or DHT with muscle fibre size or distribution. However, intramuscular pregnenolone was significantly inversely associated with muscle fibre size in both sexes and with a shift towards more type I fibres in females.
CONCLUSION:
Applying a novel LC-MS-based technique, we generated the first cross-sectional dataset on intramuscular sex hormone levels in adult males and females. Correlations between intramuscular and plasma hormone concentrations were hormone-specific, with some hormones showing strong plasma-muscle correlations whilst DHT concentrations showed no significant correlation. Furthermore, intramuscular pregnenolone confers independent associations on muscle fibre size and composition, warranting further investigation. Measuring intramuscular hormones may therefore provide novel insights into hormonal signalling at the cellular level.